Commercial breeding and egg-laying operations have long sought to identify highly fecund males that would be effective breeders. A "fecund" male is one capable of producing many offspring, as opposed to a "fertile" male, which is merely capable of producing offspring. The identification of highly fecund animals is very important to the profitable operation of commercial breeding businesses. High producing males, whose sperm reliably fertilizes female eggs, improve the productivity and profitability of breeding and laying operations. A variety of techniques have been used to improve male productivity, including artificial insemination and semen evaluation.
Aside from relying on historical data demonstrating fecundity of males, many techniques have been developed to attempt to predict fecundity of a male from examination of a seminal sample. Tests evaluating percentages of motile sperm, ion characteristics of sperm, or membrane integrity have been moderately useful but not reliably predictive of effectiveness as a breeder. Morphological examination and characterization of sperm has also been helpful, because morphologically normal sperm are more likely to be fertile. However tests evaluating these characteristics of sperm have been much more effective at distinguishing sub-fertile males from normal males, without identifying the relatively few males (top 30%) who will be the most effective breeders. Given the many morphologic, anatomic, and physiologic variables that are involved in fertilization, some experts in this field have declared that it does not appear possible to predict fertility of a male from a semen sample in the absence of historical breeding data.
The identification or isolation of motile sperm has been extensively studied in both human and veterinary medicine. The widespread adoption of assisted insemination, such as intrauterine insemination, gamete intrafallopian transfer (GIFT), or in vitro fertilization (IVF) for infertile human patients, has particularly stimulated the development of techniques for isolating, from an individual donor, a sub-population of sperm that has increased fertilizing capacity. One such assay involves layering a liquid medium on top of a semen sample, and allowing the sperm to swim-up into the medium. The sperm are then recovered and evaluated for concentration, motility and morphology. Almagor et al., J. Assisted Repro. and Gen. 10:261-265 (1993). Alternatively, sperm for in vitro fertilization can be selected by centrifuging sperm samples through gradients of Percoll solution. Chan et al., Fertil. and Steril. 61:1097-1102 (1994).
The quality of sperm from oligospermic and asthenospermic males has also been improved by allowing sperm in a seminal specimen to migrate through a tube that contains polysaccharide beads that develop rough surface ridges after hydration. Abnormal sperm are delayed from penetrating the column's pores by these ridges, which selectively allows motile sperm to be filtered out of the column. Ohashi et al., Fertil. and Steril. 57:866-870 (1992). The authors of this study reported that it was effective at slightly improving conception in infertile couples, resulting in conception in 2 out of 21 couples where the male was oligospermic.
Motile sub-populations of human sperm from an individual donor have also been isolated by suspending the sperm in a hypoosmotic medium (210 mOsm/kg) that is overlaid on an isolation medium of higher osmolality (360 mOsm/kg), that in turn overlays a recovery medium having an osmolality (290 mOsm/kg) identical to that of human uterine fluid. Motile sperm swim downward from the hypoosmotic medium toward the physiologically suitable medium (290 mOsm/kg), through interface barriers, to isolate motile sperm for IVT or GIFT. Lin et al., Arch. Androl. 27:177-184 (1991). This method is said to selectively isolate highly motile sperm from ejaculates, but it is not taught to be useful in the identification of highly fecund males.
The techniques described above are used to help improve fertilization capacity of an individual (usually oligospermic) male, instead of selecting breeding stock. Moreover, many of the findings from mammalian fertility research may not be applicable to avian breeding operations, such as poultry farms, because of substantial differences between mammalian and avian anatomy and physiology. In particular, mammalian sperm requires a biochemical activation step known as "capacitation" before fertilization can occur. Hence morphologically and functionally normal appearing mammalian sperm may be incapable of fertilization because of biochemical subtleties not measured by many morphologic and functional assays.
Fertilization in poultry also differs from mammals because spermatozoal sequestration in the oviduct's sperm-storage tubules allows a hen to lay multiple fertilized eggs after a single insemination. When a hard-shelled egg is laid, the vaginal sphincter relaxes and allows the oviduct to become momentarily patent. If sperm are sequestered in the storage tubule when the oviduct opens, fertilization may occur if sperm are released concurrently with relaxation of the sphincter, and passively transported upwardly through the oviduct by muscle contraction. These differences between mammalian and avian reproduction make it difficult to extrapolate from the results of mammalian assays, and draw reliable conclusions about the use of mammalian assays in avian species such as chickens and turkeys. Moreover, these tests have been designed to assess fertility and not fecundity.
A variety of methods have previously been proposed for the objective measurement of sperm motility in animals. In one such assay, sperm cells from a test specimen are allowed to "swim-up" into a clear medium from a concentrated sperm suspension at the bottom of an optical cuvette. Highly motile sperm cause a time-dependent increase in turbidity of the medium, which can be used to determine a fraction of rapidly moving sperm and an average velocity of the sperm. The changing turbidity of the medium is recorded by a spectrophotometer as an increase in absorbance. Sokoloski et al., Fertil. and Steril. 28:1337-1341 (1977). This reference states that no firm correlation has been established between motility and fertilizing capacity.
A swim-up technique for evaluating ram semen motility is also described in Suttiyotin and Thwaites, J. Repro. Fertil. 97:339-345 (1993). In this assay, ram semen is layered at the bottom of disposable cuvettes, and overlaid with a variety of clear media. Turbidity of the overlying medium is measured using a calorimeter, and the turbidity of the medium is said to correlate with motility. This reference does not disclose a use for this motility data.
PCT publication WO 95/29983 (Hammerstedt et al.) discloses a method for testing the potential fertility of spermatozoa in a sample by incubating an aliquot of the sperm sample with a binding protein extracted from native vitelline membranes removed from chicken or turkey eggs. The number of sperm that bind to the protein are then determined by examining the surface of a substrate on which the protein is coated. The PCT publication notes that there is a direct and linear correlation between sperm binding to the protein and the fertility of the spermatozoa in the sample. This assay is an in vitro test designed to simulate in vivo binding of the sperm to egg proteins, and the assay therefore provides an index of binding capacity of the sperm. This isolated measure of fertilization capacity does not alone provide a highly predictive measure of fertilization capacity of a male from which the sample is obtained.
In spite of years of research into the identification of highly fecund avian males, and the motivation of the commercial need for such identification, a reliable assay has not been found that predicts fecundity in males not having a breeding record. These failures have provoked pessimism among expert andrologists, who have predicted that such an assay will never be found for any species. Amman and Hammerstedt, J. Androl. 14:397-406 (1993).
It is therefore an object of this invention to provide a quick and reliable assay for the ready identification of highly fecund avian males who do not already have a breeding record.
Another object of the invention is to provide a device that is capable of performing such a method in an economical and efficient manner.
These and other objects of the invention will be better understood by reference to the following drawings and detailed description.